U.S. patent number 8,546,321 [Application Number 13/127,574] was granted by the patent office on 2013-10-01 for il-4-derived peptides for modulation of the chronic inflammatory response and treatment of autoimmune diseases.
This patent grant is currently assigned to Kobenhavns Universitet. The grantee listed for this patent is Vladimir Berezin, Elisabeth Bock. Invention is credited to Vladimir Berezin, Elisabeth Bock.
United States Patent |
8,546,321 |
Bock , et al. |
October 1, 2013 |
IL-4-derived peptides for modulation of the chronic inflammatory
response and treatment of autoimmune diseases
Abstract
The present invention relates to small peptides derived from a
cytokine, interleukin-4 (IL-4), capable of binding to the IL-4
receptors and inhibiting macrophage activation, and thereby
preventing the onset of inflammatory response. The invention
further relates to use of said peptides for the production of a
medicament for the treatment of different pathological conditions,
wherein IL-4 plays a prominent role.
Inventors: |
Bock; Elisabeth
(Charlottenlund, DK), Berezin; Vladimir (Copenhagen
N, DK) |
Applicant: |
Name |
City |
State |
Country |
Type |
Bock; Elisabeth
Berezin; Vladimir |
Charlottenlund
Copenhagen N |
N/A
N/A |
DK
DK |
|
|
Assignee: |
Kobenhavns Universitet
(DK)
|
Family
ID: |
41510516 |
Appl.
No.: |
13/127,574 |
Filed: |
November 17, 2009 |
PCT
Filed: |
November 17, 2009 |
PCT No.: |
PCT/DK2009/050304 |
371(c)(1),(2),(4) Date: |
June 14, 2011 |
PCT
Pub. No.: |
WO2010/054667 |
PCT
Pub. Date: |
May 20, 2010 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20110300148 A1 |
Dec 8, 2011 |
|
Foreign Application Priority Data
|
|
|
|
|
Nov 17, 2008 [DK] |
|
|
2008 01601 |
|
Current U.S.
Class: |
514/1.1;
514/21.5; 514/16.6; 514/21.4 |
Current CPC
Class: |
A61P
13/08 (20180101); A61P 27/16 (20180101); A61P
29/00 (20180101); A61P 11/00 (20180101); A61P
31/18 (20180101); A61P 37/06 (20180101); A61P
17/00 (20180101); A61P 25/18 (20180101); A61P
3/10 (20180101); A61P 21/00 (20180101); A61P
25/00 (20180101); A61P 7/06 (20180101); A61P
25/14 (20180101); A61P 1/00 (20180101); A61P
5/16 (20180101); A61P 15/08 (20180101); A61P
25/28 (20180101); A61P 9/00 (20180101); A61P
13/10 (20180101); A61P 37/08 (20180101); A61P
43/00 (20180101); A61P 35/00 (20180101); A61P
19/02 (20180101); A61P 37/00 (20180101); A61P
37/02 (20180101); A61P 1/16 (20180101); A61P
13/12 (20180101); A61P 9/10 (20180101); A61P
25/16 (20180101); A61P 21/04 (20180101); A61P
11/06 (20180101); C07K 14/5406 (20130101); Y02A
50/30 (20180101); A61K 38/00 (20130101); Y02A
50/414 (20180101) |
Current International
Class: |
A61K
38/00 (20060101); A61K 38/03 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
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WO 91/09059 |
|
Jun 1991 |
|
WO |
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WO 00/73460 |
|
Dec 2000 |
|
WO |
|
Other References
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Ngo et al., 1994, The Protein Folding Problem and Tertiary
Structure Prediction, Merz et al., eds., Birkhauser, Boston, pp.
492- 495. cited by examiner .
Tokuriki et al., 2009, Curr. Opin. Struc. Biol. 19:596-604. cited
by examiner .
Agnello, Davide et al., "Cytokines and Transcription Factors That
Regulate T Helper Cell Differentiation: New Players and New
Insights," Journal of Clinical Immunology, vol. 23, No. 3, May
2003, pp. 147-161. cited by applicant .
Hage, Thorsten et al., "Crystal Structure of the
Interleukin-4/Receptor .alpha. Chain Complex Reveals .alpha. Mosaic
Binding Interface," Cell, vol. 97, Apr. 1999, pp. 271-281. cited by
applicant .
He, Bei Ping et al., "Activated microglia (BV-2) facilitation of
TNF-.alpha.-mediated motor neuron death in vitro," Journal of
Neuroimmunology 128, 2002, pp. 31-38. cited by applicant .
Izuhara, K., et al., "IL-4 and IL-13 : Their Pathological Roles in
Allergic Diseases and their Potential in Developing New Therapies,"
Current Drug Targets--Inflammation & Allergy, 2002, 1, pp.
263-269. cited by applicant .
LaPorte, Sherry L., et al., "Molecular and structural basis of
cytokine receptor pleiotropy in the Interleukin-4/13 system," Cell,
2008, 132(2), pp. 259-272. cited by applicant .
Leach, Michael W., et al., "Safety Evaluation of Recombinant Human
Interleukin-4, II. Clinical Studies," Clinical Immunology and
Immunopathology, vol. 83, No. 1, 1997, pp. 12-14. cited by
applicant .
Martin, Roland, "Interleukin 4 treatment of psoriasis: are
pleiotropic cytokines suitable therapies for autoimmune diseases?"
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613-616. cited by applicant .
Martinez, Fernando Oneissi, et al., "Macrophage activation and
polarization," Frontiers in Bioscience 13, 2008, pp. 453-461. cited
by applicant .
Muller, Thomas et al., "Human Interleukin-4 and Variant R88Q:
Phasing X-ray Diffraction Data by Molecular Replacement Using X-ray
and Nuclear Magnetic Resonance Models," J. Mol. Biol. 1995, 247,
pp. 360-372. cited by applicant .
Neiiendam, Johanne Louise, et al., "An NCAM-derived FGF-receptor
agonist, the FGL-peptide, induces neurite outgrowth and neuronal
survival in primary rat neurons," Journal of Neurochemistry, 2004,
91, pp. 920-935. cited by applicant .
Ryan, Lisa K., et al., "Characterization of Proinflammatory
Cytokine Production and CD14 Expression by Murine Alveolar
Macrophage Cell Lines," In Vitro Cell, Dev. Biol.--Animal 33, 1997,
pp. 647-653. cited by applicant .
Ronn, Lars C.B., et al., "A simple procedure for quantification of
neurite outgrowth based on stereological principles," Journal of
Neuroscience Methods 100, 2000, pp. 25-32. cited by applicant .
Soroka, Vladislav et al., "Induction of Neuronal Differentiation by
a Peptide Corresponding to the Homophilic Binding Site of the
Second Ig Module of the Neural Cell Adhesion Molecule," The Journal
of Biological Chemistry, 2002, vol. 277, No. 27, pp. 24676-24683.
cited by applicant .
Szegedi, Andrea et al., "Elevated rate of Thelperl (T.sub.H1)
lymphocytes and serum IFN-.gamma. levels in psoriatic patients,"
Immunology Letters 86, 2003, pp. 277-280. cited by applicant .
Whitehead, Robert P., et al., "Phase II Trial of Recombinant Human
Interleukin-4 in Patients with Disseminated Malignant Melanoma: A
Southwest Oncology Group Study," Journal of Immunotherapy, 21,
1998, pp. 440-446. cited by applicant .
Zhang, Xinmin et al., The olfactory receptor gene superfamily of
the mouse, Nature Neuroscience, vol. 5, No. 2, 2002, pp. 124-133.
cited by applicant.
|
Primary Examiner: Kemmerer; Elizabeth C
Attorney, Agent or Firm: McLane, Graf, Raulerson &
Middleton, PA
Claims
The invention claimed is:
1. A method for treatment of inflammatory diseases or conditions
comprising administration, to an individual in need thereof, of a
compound having one or more peptides, each peptide consisting of a
sequence selected from the group consisting of TABLE-US-00005
AQFHRHKQLIRFLKRA (SEQ ID NO: 1), RNKQVIDSLAKFLKR (SEQ ID NO: 19),
ARFLKRLDRNLWGG (SEQ ID NO: 3), KRLQQNLFGG (SEQ ID NO: 6), and
Ac-AQFHRHKQLIRFLKRA (SEQ ID NO: 7),
or a variant of said sequence, said variant being substituted at 1
amino acid position with a conservative amino acid substitution,
wherein said compound is capable of decreasing LPS-induced
TNF-alpha release by macrophages.
2. A compound comprising an isolated peptide sequence consisting of
one of the following sequences TABLE-US-00006 AQFHRHKQLIRFLKRA (SEQ
ID NO: 1), RNKQVIDSLAKFLKR (SEQ ID NO: 19), ARFLKRLDRNLWGG (SEQ ID
NO: 3), KRLQQNLFGG (SEQ ID NO: 6), and Ac-AQFHRHKQLIRFLKRA (SEQ ID
NO: 7),
or a variant thereof, said variant being substituted at 1 amino
acid position with a conservative amino acid substitution, wherein
said compound is capable of decreasing LPS-induced TNF-alpha
release by macrophages.
3. A pharmaceutical composition comprising at least one compound as
defined in claim 2.
4. The method according to claim 1, wherein the inflammatory
disease or condition is an autoimmune disease or condition.
5. The method according to claim 1, wherein the inflammatory
disease or condition is rheumatoid arthritis.
6. The method according to claim 1, wherein the compound is
formulated for subcutaneous, intravenous, oral, nasal, pulmonal,
topical parenteral and/or intraarticular administration.
7. The method according to claim 1, wherein said one or more
peptides consists of a monomer of one peptide sequence.
8. The method according to claim 1, wherein said compound comprises
a multimer of two or more copies of the same peptide sequence.
9. The method according to claim 1, wherein said peptide is capable
of activating B-cells, and/or activating growth and survival of
T-cells, and/or down-regulating C5a and C3a in monocytes and
dendritic cells, and/or inhibiting macrophage activation.
10. The method according to claim 1, wherein said peptide consists
of AQFHRHKQLIRFLKRA (SEQ ID NO:1).
11. The method according to claim 1, wherein said peptide consists
of RNKQVIDSLAKFLKR (SEQ ID NO:19).
12. The method according to claim 1, wherein the inflammatory
disease or condition is ischemic heart disease.
13. The method according to claim 1, wherein said peptide consists
of ARFLKRLDRNLWGG (SEQ ID NO:3).
14. The method according to claim 1, wherein said peptide consists
of KRLQQNLFGG (SEQ ID NO:6).
15. The method according to claim 1, wherein said peptide consists
of Ac-AQFHRHKQLIRFLKRA (SEQ ID NO:7).
Description
FIELD OF INVENTION
The present invention relates to small peptides derived from a
cytokine, interleukin-4 (IL-4), capable of binding to the IL-4
receptors and inhibiting macrophage activation, and thereby
preventing the onset of inflammatory response. The invention
further relates to use of said peptides for the production of a
medicament for the treatment of different pathological conditions,
wherein IL-4 plays a prominent role.
BACKGROUND OF INVENTION
Abnormalities associated with inflammation comprise a large,
unrelated group of disorders which underlie a variety of human
diseases. Examples of disorders associated with inflammation
include asthma, chronic inflammation, and autoimmune diseases
including rheumatoid arthritis. Chronic inflammation is a
pathological condition characterised by concurrent active
inflammation, tissue destruction, and attempts at repair.
Rheumatoid arthritis (RA) is a chronic, systemic autoimmune
disorder that causes the immune system to attack the joints, where
it causes inflammation (arthritis) and destruction. It can also
damage some organs, such as the lungs and skin. It can be a
disabling and painful condition, which can lead to substantial loss
of functioning and mobility. It is diagnosed with blood tests
(especially a test called rheumatoid factor) and X-rays.
The inflammatory reaction observed in autoimmune disease involves
both cellular and soluble players. The cause of RA is not known. It
involves complex interactions of various cells, cytokines and
enzymes. The disease begins when an inciting antigen gains access
to the joint, triggering an immune response. The antigenic stimulus
activates CD4+ lymphocytes (T-cells). Once CD4+ T-cells become
activated, a complex cascade of biological events take place
including stimulation of macrophages, B-cells, fibroblasts,
chondrocytes and osteoclasts. Activated macrophages secrete
cytokines, such as interleukin-1 (IL-1), IL-6, IL-8, IL-15 and
tumor necrosis factor-.alpha. (TNF-.alpha.) (Martinez et al.,
2008).
Interleukin-4 (IL-4) is secreted by CD4+ T-cells (Th2 cells). It is
a pleiotropic cytokine, acting on various cell types and tissues.
Its action on immune cells results in activation and growth of B
cells, IgG and IgE production, MHC class II induction, growth and
survival of T cells, Th2 differentiation, enhancement of mast cell
growth, enhancement of IL-2 and IL-12-induced interferon-.gamma.
(INF-.gamma.) secretion in NK cells, downregulation of C5a and C3a
in monocytes and Mo-derived dendritic cells and inhibition of
macrophage activation (Agnello et al., 2003; Szehedi et al., 2003;
Roland, 2003).
The structure of recombinant human IL-4 has been determined by both
NMR and X-ray diffraction methods in several laboratories. It has a
classical 4 helix bundle cytokine structure (Muller et al., 1995).
IL-4, like other cytokines, exerts its biological activity by
binding to the receptors on the cell surface. One receptor complex
is composed of two components, the IL-4R .alpha. chain
(IL-4R.alpha.) and the IL-2R .gamma. chain (.gamma.c, shared by the
cytokines IL-2, IL-7, IL-9, IL-15 and IL-21), denoted type I IL-4R,
whereas the other receptor complex is composed of IL-4R.alpha. and
the IL13 .alpha. chain (IL-13R.alpha.1), called type II IL-4R. As
.gamma. c is expressed on most hematopoietic and immune cells, IL-4
is assumed to act on these cells through type I IL-4R. In contrast,
expression of IL-13R.alpha.1 is limited to some lineages such as B
cells in hematopoietic and immune cells, but ubiquitously detected
on non-immune cells (Izuhara et al., 2002). Thus IL4 acts on
non-immune cells through type II IL-4R/IL-13R.
Binding IL-4 to its receptor a chain (IL-4Ra) is a crucial event
for the generation of a Th2-dominated early immune response. The
crystal structure of the intermediate complex between human IL-4
and IL4-BP was determined at 2.3 .ANG. Resolution (PDB ID: 1IAR).
It reveals a novel spatial orientation of the two proteins, a small
but unexpected conformational change in the receptor-bound IL-4,
and an interface with three separate clusters of trans-interacting
residues (Hage et al., 1999). Crystal structure of the
Il4-Il4r-common gamma ternary complex has recently been solved (PDB
ID: 3BPL; LaPorte et al., 2008).
Recombinant IL-4 has been through several clinical trials. IL-4 has
been shown to be beneficial in patients with psoriasis, effectively
correcting imbalances in immune functions (Martin 2003). The safety
and tolerability of Escherichia coli-derived recombinant human
interleukin-4 (rhulL-4) have been evaluated in phase I and phase II
studies in human patients with a variety of malignancies. Clinical
trials have demonstrated that subcutaneous administration of
rhulL-4 is safe and well tolerated at doses as high as 5
.mu.g/kg/day and as high as 10 .mu.g/kg when administered 3
times/week. Although preclinical safety studies in cynomolgus
monkeys demonstrated a number of adverse effects following repeated
daily dosing with rhulL-4, similar effects have generally not been
observed in human patients (Leach et al., 1997). The most common
toxicities were elevated liver function tests,
nausea/vomiting/diarrhea, malaise/fatigue, edema, headache,
myalgias/arthralgias, and fever/chills. Despite promising
preclinical growth inhibitory and immunomodulatory effects, IL-4 in
this dose and schedule showed only low antitumor activity
(Whitehead et al., 1998).
Many human autoimmune and inflammatory diseases are still treated
by a combination of corticosteroids and general immunosuppression.
A better understanding of the pathogenesis of these diseases has
led to therapies that are more specific. Among these, the
recombinant humanized proteins are considered as the future
therapies. However, drugs based on recombinant proteins have
several disadvantages including high production cost, big
batch-to-batch variation and denaturation during storage.
SUMMARY OF INVENTION
The present invention concerns fragments of IL-4 that can be
chemically synthesized and used as functional mimetics of IL-4.
The present invention relates to a compound comprising an isolated
peptide consisting of at most 35 contiguous amino acid residues
derived from IL-4 or a variant being at least 70% identical. A
compound comprising such amino acid sequence is according to the
invention capable of i) binding to the IL-4 receptor; ii)
inhibiting an inflammatory response; iii) inhibiting macrophage
activation; iii) activating B-cells; iv) activating growth and
survival of T-cells; v) downregulating C5a and C3a in monocytes and
dendritic cells, vi) modulating activity of the IL-4 receptor.
Accordingly, another aspect of the invention relates to use of
compounds of the invention as medicaments and for the preparation
of medicaments for treatment of a condition or disease wherein i)
binding to the IL-4 receptor; ii) inhibiting an inflammatory
response; iii) inhibiting macrophage activation; iii) activating
B-cells; iv) activating growth and survival of T-cells; v)
downregulating C5a and C3a in monocytes and dendritic cells, vi)
modulating activity of the IL-4 receptor is part of said
treatment.
Still, in another aspect a peptide of the invention or a compound
comprising the peptide may be used for the production of an
antibody. Such antibodies will bind an epitope within a peptide of
the invention.
The invention further relates to pharmaceutical compositions
comprising a peptide of the invention, or an antibody capable of
recognising an epitope within a peptide of the invention.
The invention also concerns a method of treatment of conditions
wherein i) binding to the IL-4 receptor; ii) inhibiting an
inflammatory response; iii) inhibiting macrophage activation; iii)
activating B-cells; iv) activating growth and survival of T-cells;
v) downregulating C5a and C3a in monocytes and dendritic cells, vi)
modulating activity of the IL-4 receptor is beneficial, said method
comprising a step of administering a compound of the invention,
antibody of the invention or a pharmaceutical composition
comprising said peptide sequence, said compound or said antibody to
an individual in need.
DESCRIPTION OF DRAWINGS
FIG. 1.
Structure of IL-4 in complex with the ectodomain of IL-4R.alpha.
(PDB ID: 1IAR). Location of peptide1 (SEQ ID NO:2) (left) and
peptide3 (SEQ ID NO:3) (right) is indicated in grey.
FIG. 2.
Structure of IL-4 in complex with the ectodomain of IL-4R.alpha.
(PDB ID: 1IAR). Location of peptide3a (SEQ ID NO:1) (left) and
peptide4 (SEQ ID NO: 4) (right) is indicated in grey.
FIG. 3.
Structure of IL-4 in complex with the ectodomain of .gamma.c common
receptor (PDB ID: 3BPL). Location of peptide1 (SEQ ID NO:2) (left)
and peptide3 (SEQ ID NO:3) (right) is indicated in grey.
FIG. 4
Structure of IL-4 in complex with the ectodomain of IL-4R.alpha.
and .gamma.c common receptor (PDB ID: 3BPL). Location of peptide3a
(SEQ ID NO:1) (left) and peptide4 (SEQ ID NO: 4) (right) is
indicated in grey.
FIG. 5.
Effect of IL-4-derived peptide Ph1 (SEQ ID NO:2) on neurite
outgrowth in cultures of cerebellar granule neurons. The P2d
peptide was used a positive control (see Soroka et al., 2002).
FIG. 6.
Effect of Ph2 (SEQ ID NO:3) on neurite outgrowth in cultures of
cerebellar granule neurons. Level of significance compared to
control is represented as followed: ***=p<0.001. Seven
independent experiments were performed.
FIG. 7.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph2 (SEQ
ID NO:3).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph2 or activated by
IFN-.gamma. (striped column), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white column) or when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma. (black
column). Level of significance compared to TNF-.alpha. amount
released from non-pre-treated, activated macrophages (white column)
are represented as followed: ***=p<0.001. B: Column diagram of
the amount of TNF-.alpha. released from macrophages when
pre-treated with Ph2 in various concentrations before activation
with 0.01 .mu.g/ml IFN-.gamma.. Level of significance compared to
TNF-.alpha. amount released from non-pre-treated, activated
macrophages (0 column) is represented as followed: ***=p<0.001.
Results in both figures are shown as percentages of the untreated
control, only activated by IFN-.gamma.. Results from six
independent experiments are shown for the controls and the Ph2
concentrations 9, 27, 81 and 243 .mu.g/ml.
FIG. 8.
Binding of Ph2 (SEQ ID NO:3) to IL4r.alpha..
Binding study by applying Surface Plasmon Resonance. A: As a
control, binding between IL4 and IL4r.alpha. was investigated by
immobilizing IL4r.alpha. on a chip and then IL4 was run over the
chip in solution. B: Binding between Ph2 and IL4r.alpha. was
studied by immobilizing Ph2 on the chip and IL4r.alpha. was run
over the chip in solution. Results were analysed and KD was
calculated with the computer software BIAevaluation.
FIG. 9.
Effect of Ph3 (SEQ ID NO:1) on neurite outgrowth in cultures of
cerebellar granule neurons. Level of significance compared to
control is represented as followed: **=p<0.01. Seven independent
experiments were performed.
FIG. 10.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph3 (SEQ
ID NO:1).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph3 or activated by
IFN-.gamma. (striped column), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white column) or when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma. (black
column). Level of significance compared to TNF-.alpha. amount
released from non-pre-treated, activated macrophages (white column)
are represented as followed: ***=p<0.001. B: Column diagram of
the amount of TNF-.alpha. released from macrophages when
pre-treated with Ph3 in various concentrations before activation
with 0.01 .mu.g/ml IFN-.gamma.. Level of significance compared to
TNF-.alpha. amount released from non-pre-treated, activated
macrophages (0 column) is represented as followed: ***=p<0.001.
Results in both figures are shown as percentages of the untreated
control, only activated by IFN-.gamma.. Results from six
independent experiments are shown for the controls and the Ph3
concentrations 9, 27, and 81 .mu.g/ml.
FIG. 11.
Binding of Ph3 (SEQ ID NO:1) to IL4r.alpha..
Binding study by applying Surface Plasmon Resonance. A: As a
control, binding between IL4 and IL4r.alpha. was investigated by
immobilizing IL4r.alpha. on a chip and then IL4 was run over the
chip in solution. B: Binding between Ph3 and IL4r.alpha. was
studied by immobilizing Ph3 on the chip and IL4r.alpha. was run
over the chip in solution. Results were analysed and KD was
calculated with the computer software BIAevaluation.
FIG. 12.
Effect of Ph4 (SEQ ID NO:4) on neurite outgrowth in cultures of
cerebellar granule neurons. Level of significance compared to
control is represented as followed: *=p<0.05, **=p<0.01. Five
independent experiments were performed.
FIG. 13.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph5 (SEQ
ID NO:5).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph4 or activated by
IFN-.gamma. (stripes), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white) and when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma.
(black). B: Column diagram of the amount of TNF-.alpha. released
from macrophages when pre-treated with 9 .mu.g/ml Ph5 before
activation with 0.01 .mu.g/ml IFN-.gamma.. Two independent
experiments were performed.
FIG. 14.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph6 (SEQ
ID NO:6).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph4 or activated by
IFN-.gamma. (stripes), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white) and when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma.
(black). B: Column diagram of the amount of TNF-.alpha. released
from macrophages when pre-treated with various concentrations of
Ph6 before activation with 0.01 .mu.g/ml IFN-.gamma.. Two
independent experiments were performed.
FIG. 15.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph8 (SEQ
ID NO:1).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph3 or activated by
IFN-.gamma. (striped column), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white column) or when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma. (black
column). Level of significance compared to TNF-.alpha. amount
released from non-pre-treated, activated macrophages (white column)
are represented as followed: ***=p<0.001. B: Column diagram of
the amount of TNF-.alpha. released from macrophages when
pre-treated with Ph8 in various concentrations before activation
with 0.01 .mu.g/ml IFN-.gamma.. Level of significance compared to
TNF-.alpha. amount released from non-pre-treated, activated
macrophages (0 column) is represented as followed: ***=p<0.001.
Results in both figures are shown as percentages of the untreated
control, only activated by IFN-.gamma.. Results from six
independent experiments are shown for the controls and the Ph8
concentrations 9, 27, 81 and 243 .mu.g/ml.
FIG. 16.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph10 (SEQ
ID:1).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph10 or activated by
IFN-.gamma. (striped column), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white column) or when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma. (black
column). Level of significance compared to TNF-.alpha. amount
released from non-pre-treated, activated macrophages (white column)
are represented as followed: **=p<0.01. B: Column diagram of the
amount of TNF-.alpha. released from macrophages when pre-treated
with Ph10 in various concentrations before activation with 0.01
.mu.g/ml IFN-.gamma.. Level of significance compared to TNF-.alpha.
amount released from non-pre-treated, activated macrophages (0
column) is represented as followed: **=p<0.01. Results in both
figures are shown as percentages of the untreated control, only
activated by IFN-.gamma.. Results from four independent experiments
are shown for the controls and the Ph10 concentrations 9, 27, 81
and 243 .mu.g/ml. Only two experiments were performed with the
concentration 54 .mu.g/ml Ph10 which does that these data were not
included in the statistical analysis.
FIG. 17.
Macrophage secretion of TNF-.alpha. when pre-treated with Ph12 (SEQ
ID:19).
A: Column diagram of the amount of TNF-.alpha. released from
macrophages when not pre-treated with Ph12 or activated by
IFN-.gamma. (stripes), when activated with 0.01 .mu.g/ml
IFN-.gamma. (white) and when pre-treated with 100 .mu.M
hydrocortisone and activated with 0.01 .mu.g/ml IFN-.gamma.
(black). B: Column diagram of the amount of TNF-.alpha. released
from macrophages when pre-treated with Ph12 in various
concentrations before activation with 0.01 .mu.g/ml IFN-.gamma..
Two independent experiments were performed.
DETAILED DESCRIPTION OF THE INVENTION
A compound according to the invention can be a fragment derived
from interleukin-4, or it may be derived from a variant of
interleukin-4, such as a natural or recombinant interleukin-4
variant, for example a interleukin-4 variant produced by
alternative splicing, or genetic polymorphism, or any type of
recombinant interleukin-4.
A peptide according to the invention is a peptide which is capable
of interacting with the IL-4 receptor, modulating IL-4 receptor
signalling, activating B-cells, activating growth and survival of
T-cells, downregulating C5a and C3a in monocytes and dendritic
cells or inhibiting macrophage activation.
By the terms "modulation" or "modulating" are meant a change, such
as an inhibition or stimulation. By the term "interacting" is meant
an action, such as binding, between the peptide and the IL-4
receptor which cause an effect.
Amino Acid Sequence
Compounds according to the invention comprise a peptide consisting
of a contiguous amino acid sequence derived from IL-4 or a fragment
or variant thereof.
In one embodiment the compound according to the invention may
comprise a peptide consisting of at most 35 contiguous amino acids
which is derived from interleukin-4 (SEQ ID:38) or a fragment
thereof, or a variant being at least 70% identical to SEQ ID NO:38
or a fragment thereof.
The amino acid sequence of the human IL-4 precursor (Swiss-Prot ID:
P05112) is:
TABLE-US-00001 (SEQ ID NO: 38) MGLTSQLLPP LFFLLACAGN FVHGHKCDIT
LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL
GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK
CSS
A peptide sequence according to the invention consists of at most
35 contiguous amino acid residues, such as from 3 to 35 amino acid
residues, such as from 3 to 30, for example from 3 to 25, such as
from 5 to 25, such as form 7 to 25, such as from 8 to 25, for
example from 10 to 25, or from 12 to 25, such as from 14 to 25.
Sequences comprising from 5 to 25 contiguous amino acid residues
are preferred.
In a preferred embodiment said peptides of the invention comprise
at most 35 contiguous amino acids which are derived from an
alpha-helix of IL-4.
By the term "alpha-helix" is meant the common motif in the
secondary structure of proteins, the alpha helix (.alpha.-helix) is
a right- or left-handed coiled conformation, in which every
backbone N--H group donates a hydrogen bond to the backbone C.dbd.O
group of the amino acid four residues earlier.
In a preferred embodiment said peptides of the invention comprise a
sequence with the formula X1-X2-X3, wherein X1 is L, X2 is I, Q, G,
T, or a charged amino acid; and X3 is Q, T, or a charged amino
acid.
In one preferred embodiment X2 is I or Q.
In a more preferred embodiment X2 is I.
In another more preferred embodiment X2 is Q.
In one preferred embodiment X2 is a charged amino acid.
In a preferred embodiment X3 is a charged amino acid.
In a more preferred embodiment X3 is R or E.
In one most preferred embodiment X3 is R.
In another more preferred embodiment X3 is E.
In another preferred embodiment X3 is Q or T.
In an even more preferred embodiment X1 is L, X2 is I, and X3 is
R.
In another even more preferred embodiment X1 is L, X2 is Q and X3
is E.
In a most preferred embodiment said peptides of the invention
consist of an amino acid sequence selected from one of the
following amino acid sequences:
TABLE-US-00002 AQFHRHKQLIRFLKRA SEQ ID NO: 1 AITLQEIIKTLNSA SEQ ID
NO: 2 ARFLKRLDRNLWGG SEQ ID NO: 3 AERLKTIMREKYSKS SEQ ID NO: 4
LQEIKTLN SEQ ID NO: 5 KRLQQNLFGG SEQ ID NO: 6 Ac-AQFHRHKQLIRFLKRA
SEQ ID NO: 7 QEIIKKL SEQ ID NO: 8 AIQNQEEIKYLNS SEQ ID NO: 9
AIILQEI SEQ ID NO: 10 IVLQEII SEQ ID NO: 11 TLGEIIKGVNS SEQ ID NO:
12 VTLIDHSEEIFKTLN SEQ ID NO: 13 LQERIKSLN SEQ ID NO: 14
RLDRENVAVYNLW SEQ ID NO: 15 LRSLDRNL SEQ ID NO: 16 RLLRLDRN SEQ ID
NO: 17 RFLKRYFYNLEENL SEQ ID NO: 18 RNKQVIDSLAKFLKR SEQ ID NO: 19
RHKALIR SEQ ID NO: 20 KKLIRYLK SEQ ID NO: 21 RHKTLIR SEQ ID NO: 22
MQDKYSKS SEQ ID NO: 23 AERVKIEQREYKKYS SEQ ID NO: 24 SQLIRFLKRLA
SEQ ID NO: 25 TVTDIFAASKNTT SEQ ID NO: 26 TLENFLERLKTA SEQ ID NO:
27 TEKEVLRQFYSA SEQ ID NO: 28 KTLTELTKTLNS SEQ ID NO: 29
AHKEIIKTLNSLQKA SEQ ID NO: 30 AKTLSTELTVTA SEQ ID NO: 31
STLENFLERLA SEQ ID NO: 32 NEERLKTIMRA SEQ ID NO: 33 RAATVLRQFYSR
SEQ ID NO: 34 KTLNSLTEQKT SEQ ID NO: 35 AHRHKQLIRA SEQ ID NO: 36
ATAQQFHRHKQA SEQ ID NO: 37
or a variant or fragment thereof.
In one embodiment the said peptides of the invention consist of an
amino acid sequence selected from one of the following amino acid
sequences:
TABLE-US-00003 AQFHRHKQLIRFLKRA (SEQ ID NO: 1) Ac-AQFHRHKQLIRFLKRA
(SEQ ID NO: 7) RHKALIR (SEQ ID NO: 20) KKLIRYLK (SEQ ID NO: 21)
RHKTLIR (SEQ ID NO: 22) SQLIRFLKRLA (SEQ ID NO: 25) AHRHKQLIRA (SEQ
ID NO: 36)
or a variant or fragment thereof.
In one embodiment the said peptides of the invention consist of an
amino acid sequence selected from one of the following amino acid
sequences:
TABLE-US-00004 AITLQEIIKTLNSA (SEQ ID NO: 2) LQEIKTLN (SEQ ID NO:
5) AIILQEI (SEQ ID NO: 10) IVLQEII (SEQ ID NO: 11) LQERIKSLN (SEQ
ID NO: 14) AHKEIIKTLNSLQKA (SEQ ID NO: 30)
or a variant or fragment thereof.
In the present context the standard one-letter code for amino acid
residues as well as the standard three-letter code are applied.
Abbreviations for amino acids are in accordance with the
recommendations in the IUPAC-IUB Joint Commission on Biochemical
Nomenclature Eur. J. Biochem, 1984, vol. 184, pp 9-37. Throughout
the description and claims either the three letter code or the one
letter code for natural amino acids are used. Where the L or D form
has not been specified it is to be understood that the amino acid
in question has the natural L form, cf. Pure & Appl. Chem. Vol.
(56(5) pp 595-624 (1984) or the D form, so that the peptides formed
may be constituted of amino acids of L form, D form, or a sequence
of mixed L forms and D forms.
Where nothing is specified it is to be understood that the
C-terminal amino acid of a peptide for use according to the
invention exists as the free carboxylic acid, this may also be
specified as "--OH". However, the C-terminal amino acid of a
peptide for use according to the invention may be the amidated
derivative, which is indicated as "--NH.sub.2". Where nothing else
is stated the N-terminal amino acid of a polypeptide comprises a
free amino-group, this may also be specified as "H--".
A peptide, fragment or variant thereof according to the invention
can also comprise one or several unnatural amino acids.
A preferred peptide according to the invention is an isolated
contiguous peptide sequence which comprises at most 35 amino acid
residues of IL-4. It is understood that all peptides according to
the invention comprise at least one amino acid sequence selected
from any of the sequences SEQ ID NOs: 1-37 or a fragment or variant
thereof.
Thus, some embodiments of the invention may relate to a peptide
comprising a fragment of a sequence selected from SEQ ID NOs:1 to
37. Another embodiment may relate to variants of SEQ ID
NOs:1-37.
In one embodiment a variant fragment varies compared to a fragment
of SEQ ID NO 38. A variant fragment may differ from a fragment of
SEQ ID NO 38 by having a different amino acid at one or more
positions. Preferably the variant differs from the fragment of SEQ
ID NO 38 at up to 10 amino acid positions, more preferably at up to
8 position, such as up to 6 positions, for example up to 5
positions, such as at 4, 3, 2 or 1 position. Such variants may also
differ from a fragment of SEQ ID NO 38 in other ways, such as by
having one or more chemical modifications.
A variant according to the invention of an amino acid sequence
selected from the sequences SEQ ID NOs: 1-38 may be i) an amino
acid sequence which has at least 70% identity with a selected
sequence, such as 71-75% identity, for example 76-80% identity,
such as 81-85% identity, such as 86-90% identity, for example
91-95% identity, such as 96-99% identity, wherein the identity is
defined as a percentage of identical amino acids in said sequence
when it is collated with the selected sequence. The identity
between amino acid sequences may be calculated using well known
algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50,
BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75,
BLOSUM 80, BLOSUM 85, or BLOSUM 90; ii) an amino acid sequence
which has at least 70% positive amino acid matches with a selected
sequence, such as 71-80% positive amino acid matches, for example
81-85% positive amino acid matches, such as 86-90% positive amino
acid matches, for example 91-95% positive amino acid matches, such
as 96-99% positive amino acid matches, wherein the positive amino
acid match is defined as the presence at the same position in two
compared sequences of amino acid residues which has similar
physical and/or chemical properties. Preferred positive amino acid
matches of the present invention are K to R, E to D, L to M, Q to
E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S and Q to
R; iii) an amino acid sequence which is identical to a selected
sequence, or it has at least 70% identity with said sequence such
as 71-80% identity, for example 81-85% identity, such as 86-90%
identity, for example 91-95% identity, such as 96-99% identity, or
has at least 75% positive amino acid matches with the selected
sequence, such as 76-80% positive amino acid matches, for example
81-85% positive amino acid matches, such as 86-90% positive amino
acid matches, for example 91-95% positive amino acid matches, such
as 96-99% positive amino acid matches, and comprises other chemical
moieties, e.g. phosphoryl, sulphur, acetyl, glycosyl moieties.
The term "variant of a peptide sequence" also means that the
peptide sequence may be modified, for example by substitution of
one or more of the amino acid residues. Both L-amino acids and
D-amino acids may be used. Other modification may comprise
derivatives such as esters, sugars, etc., for example methyl and
acetyl esters, as well as polyethylene glycol modifications.
Furthermore, an amine group of the peptide may be converted to
amides, wherein the acid part of the amide is a fatty acid.
In another aspect, variants of the amino acid sequences according
to the invention may comprise, within the same variant, or
fragments thereof or among different variants, or fragments
thereof, at least one substitution, such as a plurality of
substitutions introduced independently of one another. Variants of
the complex, or fragments thereof may thus comprise conservative
substitutions independently of one another, wherein at least one
glycine (Gly) of said variant, or fragments thereof is substituted
with an amino acid selected from the group of amino acids
consisting of Ala, Val, Leu, and Ile, and independently thereof,
variants, or fragments thereof, wherein at least one alanine (Ala)
of said variants, or fragments thereof is substituted with an amino
acid selected from the group of amino acids consisting of Gly, Val,
Leu, and Ile, and independently thereof, variants, or fragments
thereof, wherein at least one valine (Val) of said variant, or
fragments thereof is substituted with an amino acid selected from
the group of amino acids consisting of Gly, Ala, Leu, and Ile, and
independently thereof, variants, or fragments thereof, wherein at
least one leucine (Leu) of said variant, or fragments thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Gly, Ala, Val, and Ile, and independently
thereof, variants, or fragments thereof, wherein at least one
isoleucine (Ile) of said variants, or fragments thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Gly, Ala, Val and Leu, and independently
thereof, variants, or fragments thereof wherein at least one
aspartic acids (Asp) of said variant, or fragments thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Glu, Asn, and Gin, and independently thereof,
variants, or fragments thereof, wherein at least one aspargine
(Asn) of said variants, or fragments thereof is substituted with an
amino acid selected from the group of amino acids consisting of
Asp, Glu, and Gin, and independently thereof, variants, or
fragments thereof, wherein at least one glutamine (Gln) of said
variants, or fragments thereof is substituted with an amino acid
selected from the group of amino acids consisting of Asp, Glu, and
Asn, and wherein at least one phenylalanine (Phe) of said variants,
or fragments thereof is substituted with an amino acid selected
from the group of amino acids consisting of Tyr, Trp, His, Pro, and
preferably selected from the group of amino acids consisting of Tyr
and Trp, and independently thereof, variants, or fragments thereof,
wherein at least one tyrosine (Tyr) of said variants, or fragments
thereof is substituted with an amino acid selected from the group
of amino acids consisting of Phe, Trp, His, Pro, preferably an
amino acid selected from the group of amino acids consisting of Phe
and Trp, and independently thereof, variants, or fragments thereof,
wherein at least one arginine (Arg) of said fragment is substituted
with an amino acid selected from the group of amino acids
consisting of Lys and His, and independently thereof, variants, or
fragments thereof, wherein at least one lysine (Lys) of said
variants, or fragments thereof is substituted with an amino acid
selected from the group of amino acids consisting of Arg and His,
and independently thereof, variants, or fragments thereof, and
independently thereof, variants, or fragments thereof, and wherein
at least one proline (Pro) of said variants, or fragments thereof
is substituted with an amino acid selected from the group of amino
acids consisting of Phe, Tyr, Trp, and His, and independently
thereof, variants, or fragments thereof, wherein at least one
cysteine (Cys) of said variants, or fragments thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr,
and Tyr.
It thus follows from the above that the same variant of a peptide
fragment, or fragment of said variant may comprise more than one
conservative amino acid substitution from more than one group of
conservative amino acids as defined herein above. The term
"conservative amino acid substitution" is used synonymously herein
with the term "homologous amino acid substitution".
The groups of conservative amino acids are as the following:
A, G (neutral, weakly hydrophobic),
Q, N, S, T (hydrophilic, non-charged)
E, D (hydrophilic, acidic)
H, K, R (hydrophilic, basic)
L, P, I, V, M, F, Y, W (hydrophobic, aromatic)
C (cross-link forming)
Conservative substitutions may be introduced in any position of a
preferred predetermined peptide for use according to the invention
or fragment thereof. It may however also be desirable to introduce
non-conservative substitutions, particularly, but not limited to, a
non-conservative substitution in any one or more positions.
A non-conservative substitution leading to the formation of a
variant fragment of the peptide for use according to the invention
would for example differ substantially in polarity, for example a
residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile,
Leu, Phe or Met) substituted for a residue with a polar side chain
such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino
acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a
polar residue for a non-polar one; and/or ii) differ substantially
in its effect on peptide backbone orientation such as substitution
of or for Pro or Gly by another residue; and/or iii) differ
substantially in electric charge, for example substitution of a
negatively charged residue such as Glu or Asp for a positively
charged residue such as Lys, His or Arg (and vice versa); and/or
iv) differ substantially in steric bulk, for example substitution
of a bulky residue such as His, Trp, Phe or Tyr for one having a
minor side chain, e.g. Ala, Gly or Ser (and vice versa).
Substitution of amino acids may in one embodiment be made based
upon their hydrophobicity and hydrophilicity values and the
relative similarity of the amino acid side-chain substituents,
including charge, size, and the like.
A peptide according to the invention is a peptide which is capable
of interacting with the IL-4 receptor.
In one embodiment the peptide according to the invention is capable
of modulating IL-4 receptor signalling.
In a preferred embodiment the peptide according to the invention is
capable of stimulating IL-4 signalling. In another preferred
embodiment the peptide according to the invention is capable of
inhibiting IL-4 receptor signalling.
In another embodiment the peptide according to the invention is
capable of activating B-cells.
In a further embodiment the peptide according to the invention is
capable of activating growth and survival of T-cells.
In another embodiment the peptide according to the invention is
capable of downregulating C5a and C3a in monocytes and dendritic
cells.
In yet another embodiment the peptide according to the invention is
capable of inhibiting macrophage activation.
Both fragments and variants of amino acid sequences according to
the invention are functional equivalents of said sequences.
By the term "functional equivalent" of an amino acid sequence is in
the present context meant a molecule which meets the criteria for a
variant or a fragment of said amino acid sequence described above
and which is capable of one or more functional activities of said
sequence or a compound comprising said sequence. In a preferred
embodiment, the functional equivalent of an amino acid sequence
according to the invention, is capable of interacting with the IL-4
receptor and modulate IL-4 receptor signalling.
The invention relates both to isolated peptides according to the
invention and fusion proteins comprising peptides according to the
invention.
In one embodiment, the peptide according to the invention is an
isolated peptide. By the term "isolated peptide" is meant that the
peptide according to the invention is an individual compound and
not a part of another compound. The isolated peptide may be
produced by use of any recombinant technology methods or chemical
synthesis and separated from other compounds, or it may be
separated from a longer polypeptide or protein by a method of
enzymatic or chemical cleavage and further separated from other
protein fragments.
The peptide sequence may be present in the compound as a single
copy, i.e. formulated as a monomer of the peptide sequence, or it
may be present as several copies of the same sequence, e.g. as a
multimer comprising two or more copies of a sequence selected from
SEQ ID NOs:1-37, or two or more copies of a fragment or a variant
of said sequence.
An isolated peptide according to the invention may in another
embodiment comprise a fragment of interleukin-4 which consists of a
contiguous amino acid sequence derived from interleukin-4, selected
from SEQ ID NOs:1-37 or a variant thereof. In another embodiment
the isolated peptide may consist of one or more of the sequences
SEQ ID NOs:1-37.
Production of Peptide Sequences
The peptide sequences of the present invention may be prepared by
any conventional synthetic methods, recombinant DNA technologies,
enzymatic cleavage of full-length proteins which the peptide
sequences are derived from, or a combination of said methods.
Synthetic Preparation
The methods for synthetic production of peptides are well known in
the art. Detailed descriptions as well as practical advice for
producing synthetic peptides may be found in Synthetic Peptides: A
User's Guide (Advances in Molecular Biology), Grant G. A. ed.,
Oxford University Press, 2002, or in: Pharmaceutical Formulation:
Development of Peptides and Proteins, Frokjaer and Hovgaard eds.,
Taylor and Francis, 1999.
Peptides may for example be synthesised by using Fmoc chemistry and
with Acm-protected cysteins. After purification by reversed phase
HPLC, peptides may be further processed to obtain for example
cyclic or C- or N-terminal modified isoforms. The methods for
cyclization and terminal modification are well-known in the art and
described in detail in the above-cited manuals.
In a preferred embodiment the peptide sequences of the invention
are produced synthetically, in particular, by the Sequence Assisted
Peptide Synthesis (SAPS) method.
Peptides may be synthesised either batchwise in a polyethylene
vessel equipped with a polypropylene filter for filtration or in
the continuous-flow version of the polyamide solid-phase method
(Dryland, A. and Sheppard, R. C., (1986) J. Chem. Soc. Perkin
Trans. I, 125-137.) on a fully automated peptide synthesiser using
9-fluorenylmethyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl,
(Boc) as N-a-amino protecting group and suitable common protection
groups for side-chain functionality's.
Recombinant Preparation
Thus, in one embodiment the peptides of the invention are produced
by use of recombinant DNA technologies.
The DNA sequence encoding a peptide or the corresponding
full-length protein the peptide originates from may be prepared
synthetically by established standard methods, e.g. the
phosphoamidine method described by Beaucage and Caruthers, 1981,
Tetrahedron Lett. 22:1859-1869, or the method described by Matthes
et al., 1984, EMBO J. 3:801-805. According to the phosphoamidine
method, oligonucleotides are synthesised, e.g. in an automatic DNA
synthesiser, purified, annealed, ligated and cloned in suitable
vectors.
The DNA sequence encoding a peptide may also be prepared by
fragmentation of the DNA sequences encoding the corresponding
full-length protein of peptide origin, using DNAase I according to
a standard protocol (Sambrook et al., Molecular cloning: A
Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, N.Y.,
1989). The present invention relates to full-length proteins
selected from the groups of proteins identified above. The DNA
encoding the full-length proteins of the invention may
alternatively be fragmented using specific restriction
endonucleases. The fragments of DNA are further purified using
standard procedures described in Sambrook et al., Molecular
cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring
Harbor, N.Y., 1989.
The DNA sequence encoding a full-length protein may also be of
genomic or cDNA origin, for instance obtained by preparing a
genomic or cDNA library and screening for DNA sequences coding for
all or part of the full-length protein by hybridisation using
synthetic oligonucleotide probes in accordance with standard
techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor, 1989). The DNA sequence may
also be prepared by polymerase chain reaction using specific
primers, for instance as described in U.S. Pat. No. 4,683,202 or
Saiki et al., 1988, Science 239:487-491.
The DNA sequence is then inserted into a recombinant expression
vector, which may be any vector, which may conveniently be
subjected to recombinant DNA procedures. The choice of vector will
often depend on the host cell into which it is to be introduced.
Thus, the vector may be an autonomously replicating vector, i.e. a
vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one which, when introduced into a
host cell, is integrated into the host cell genome and replicated
together with the chromosome(s) into which it has been
integrated.
In the vector, the DNA sequence encoding a peptide or a full-length
protein should be operably connected to a suitable promoter
sequence. The promoter may be any DNA sequence, which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of the coding DNA sequence in mammalian
cells are the SV 40 promoter (Subramani et al., 1981, Mol. Cell
Biol. 1:854-864), the MT-1 (metallothionein gene) promoter
(Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2
major late promoter. A suitable promoter for use in insect cells is
the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.
311:7-11). Suitable promoters for use in yeast host cells include
promoters from yeast glycolytic genes (Hitzeman et al., 1980, J.
Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol.
Appl. Gen. 1: 419-434) or alcohol dehydrogenase genes (Young et
al., 1982, in Genetic Engineering of Microorganisms for Chemicals,
Hollaender et al, eds., Plenum Press, New York), or the TPI1 (U.S.
Pat. No. 4,599,311) or ADH2-4c (Russell et al., 1983, Nature
304:652-654) promoters. Suitable promoters for use in filamentous
fungus host cells are, for instance, the ADH3 promoter (McKnight et
al., 1985, EMBO J. 4:2093-2099) or the tpiA promoter.
The coding DNA sequence may also be operably connected to a
suitable terminator, such as the human growth hormone terminator
(Palmiter et al., op. cit.) or (for fungal hosts) the TPI1 (Alber
and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.)
promoters. The vector may further comprise elements such as
polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb
region), transcriptional enhancer sequences (e.g. the SV 40
enhancer) and translational enhancer sequences (e.g. the ones
encoding adenovirus VA RNAs).
The recombinant expression vector may further comprise a DNA
sequence enabling the vector to replicate in the host cell in
question. An example of such a sequence (when the host cell is a
mammalian cell) is the SV 40 origin of replication. The vector may
also comprise a selectable marker, e.g. a gene the product of which
complements a defect in the host cell, such as the gene coding for
dihydrofolate reductase (DHFR) or one which confers resistance to a
drug, e.g. neomycin, hydromycin or methotrexate.
The procedures used to ligate the DNA sequences coding the peptides
or full-length proteins, the promoter and the terminator,
respectively, and to insert them into suitable vectors containing
the information necessary for replication, are well known to
persons skilled in the art (cf., for instance, Sambrook et al., op.
cit.).
To obtain recombinant peptides of the invention the coding DNA
sequences may be usefully fused with a second peptide coding
sequence and a protease cleavage site coding sequence, giving a DNA
construct encoding the fusion protein, wherein the protease
cleavage site coding sequence positioned between the HBP fragment
and second peptide coding DNA, inserted into a recombinant
expression vector, and expressed in recombinant host cells. In one
embodiment, said second peptide selected from, but not limited by
the group comprising glutathion-S-reductase, calf thymosin,
bacterial thioredoxin or human ubiquitin natural or synthetic
variants, or peptides thereof. In another embodiment, a peptide
sequence comprising a protease cleavage site may be the Factor Xa,
with the amino acid sequence IEGR, enterokinase, with the amino
acid sequence DDDDK, thrombin, with the amino acid sequence
LVPR/GS, or Acharombacter lyticus, with the amino acid sequence
XKX, cleavage site.
The host cell into which the expression vector is introduced may be
any cell which is capable of expression of the peptides or
full-length proteins, and is preferably a eukaryotic cell, such as
invertebrate (insect) cells or vertebrate cells, e.g. Xenopus
laevis oocytes or mammalian cells, in particular insect and
mammalian cells. Examples of suitable mammalian cell lines are the
HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632,
ATCC CCL-10) or CHO (ATCC CCL-61) cell lines. Methods of
transfecting mammalian cells and expressing DNA sequences
introduced in the cells are described in e.g. Kaufman and Sharp, J.
Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J. Mol.
Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.
USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and
Pearson, 1981, in Somatic Cell Genetics 7, p. 603; Graham and van
der Eb, 1973, Virol. 52:456; and Neumann et al., 1982, EMBO J.
1:841-845.
Alternatively, fungal cells (including yeast cells) may be used as
host cells. Examples of suitable yeast cells include cells of
Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae. Examples of other fungal cells
are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora
spp., in particular strains of Aspergillus oryzae or Aspergillus
niger. The use of Aspergillus spp. for the expression of proteins
is described in, e.g., EP 238 023.
The medium used to culture the cells may be any conventional medium
suitable for growing mammalian cells, such as a serum-containing or
serum-free medium containing appropriate supplements, or a suitable
medium for growing insect, yeast or fungal cells. Suitable media
are available from commercial suppliers or may be prepared
according to published recipes (e.g. in catalogues of the American
Type Culture Collection).
The peptides or full-length proteins recombinantly produced by the
cells may then be recovered from the culture medium by conventional
procedures including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. HPLC, ion exchange chromatography, affinity
chromatography, or the like.
Medicament
It is an objective of the invention to provide a compound capable
of modulating the activity of IL-4, said compound according to the
invention can be used as a medicament for the treatment of
diseases, wherein modulation of IL-4 signalling may be considered
as an essential condition for curing.
Accordingly, the invention relates to the use of one or more of the
peptides comprising a sequence derived from IL-4 or a fragment or
variant thereof for the manufacture of a medicament.
In one embodiment the medicament of the invention comprises at
least one of the amino acid sequences set forth in SEQ ID NOS: 1-37
or fragments or variants of said sequences. In another embodiment
the medicament of the invention comprises an antibody capable of
binding to an epitope in IL-4 or a fragment thereof or a fragment
or variant of said antibody.
The medicament of the invention comprises an effective amount of
one or more of the compounds as defined above, or a composition
comprising a compound as defined above, in combination with
pharmaceutically acceptable additives. Such medicament may suitably
be formulated for oral, percutaneous, subcutaneous, topical,
intramuscular, intravenous, intracranial, intrathecal,
intracerebroventricular, nasal, intranasal or pulmonal
administration or parental administration supplemented with
intraarticular administration into or near joint capsules.
Strategies in formulation development of medicaments and
compositions based on the peptides of the present invention
generally correspond to formulation strategies for any other
protein-based drug product. Potential problems and the guidance
required to overcome these problems are dealt with in several
textbooks, e.g. "Therapeutic Peptides and Protein Formulation.
Processing and Delivery Systems", Ed. A. K. Banga, Technomic
Publishing AG, Basel, 1995.
Injectables are usually prepared either as liquid solutions or
suspensions, solid forms suitable for solution in, or suspension
in, liquid prior to injection. The preparation may also be
emulsified. The active ingredient is often mixed with excipients
which are pharmaceutically acceptable and compatible with the
active ingredient. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol or the like, and combinations
thereof. In addition, if desired, the preparation may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, or which enhance the effectiveness or
transportation of the preparation.
Formulations of the compounds of the invention can be prepared by
techniques known to the person skilled in the art. The formulations
may contain pharmaceutically acceptable carriers and excipients
including microspheres, liposomes, microcapsules, nanoparticles or
the like.
The preparation may suitably be administered by injection,
optionally at the site, where the active ingredient is to exert its
effect. Additional formulations which are suitable for other modes
of administration include suppositories, nasal, pulmonal and, in
some cases, oral formulations. For suppositories, traditional
binders and carriers include polyalkylene glycols or triglycerides.
Such suppositories may be formed from mixtures containing the
active ingredient(s) in the range of from 0.5% to 10%, preferably
1-2%. Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders and generally contain 10-95% of the active
ingredient(s), preferably 25-70%.
Other formulations are such suitable for nasal and pulmonal
administration, e.g. inhalators and aerosols.
The active compound may be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include acid addition salts (for
example formed with the free amino groups of the peptide compound)
and which are formed with inorganic acids such as, for example,
hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric
acids and the like, or such organic acids as formic, acetic,
trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic,
citric, fumaric, glycolic, lactic, maleic, malic, malonic,
mandelic, oxalic, picric, pyruvic, salicylic, succinic,
methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic,
bismethylene salicylic, ethanedisulfonic, gluconic, citraconic,
aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic,
glutamic, benzenesulfonic, p-toluenesulfonic acids and the like.
Salts formed with the free carboxyl group may also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
procaine, and the like.
Further examples of pharmaceutically acceptable inorganic or
organic acid addition salts include the pharmaceutically acceptable
salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated
herein by reference. Examples of metal salts include lithium,
sodium, potassium, magnesium salts and the like. Examples of
ammonium and alkylated ammonium salts include ammonium,
methylammonium, dimethylammonium, trimethylammonium, ethylammonium,
hydroxyethylammonium, diethylammonium, butylammonium,
tetramethylammonium salts and the like.
The preparations are administered in a manner compatible with the
dosage formulation, and in such amount as will be therapeutically
effective. The quantity to be administered depends on the subject
to be treated, including, e.g. the weight and age of the subject,
the disease to be treated and the stage of disease. Suitable dosage
ranges are per kilo body weight normally of the order of several
hundred .mu.g active ingredient per administration with a preferred
range of from about 0.1 .mu.g to 5000 .mu.g per kilo body weight.
Using monomeric forms of the compounds, the suitable dosages are
often in the range of from 0.1 .mu.g to 5000 .mu.g per kilo body
weight, such as in the range of from about 0.1 .mu.g to 3000 .mu.g
per kilo body weight, and especially in the range of from about 0.1
.mu.g to 1000 .mu.g per kilo body weight. Using multimeric forms of
the compounds, the suitable dosages are often in the range of from
0.1 .mu.g to 1000 .mu.g per kilo body weight, such as in the range
of from about 0.1 .mu.g to 750 .mu.g per kilo body weight, and
especially in the range of from about 0.1 .mu.g to 500 .mu.g per
kilo body weight such as in the range of from about 0.1 .mu.g to
250 .mu.g per kilo body weight. In particular when administering
nasally smaller dosages are used than when administering by other
routes. Administration may be performed once or may be followed by
subsequent administrations. The dosage will also depend on the
route of administration and will vary with the age and weight of
the subject to be treated. A preferred dosage of multimeric forms
would be in the interval 1 mg to 70 mg per 70 kg body weight.
For most indications a localised or substantially localised
application is preferred.
Some of the compounds of the present invention are sufficiently
active, but for some of the others, the effect will be enhanced if
the preparation further comprises pharmaceutically acceptable
additives and/or carriers. Such additives and carriers will be
known in the art. In some cases, it will be advantageous to include
a compound, which promotes delivery of the active substance to its
target.
In many instances, it will be necessary to administrate the
formulation multiple times. Administration may be a continuous
infusion, such as intraventricular infusion or administration in
more doses such as more times a day, daily, more times a week,
weekly, etc. It is preferred that administration of the medicament
is initiated before or shortly after the individual has been
subjected to the factor(s) that may lead to cell death. Preferably
the medicament is administered within 8 hours from the factor
onset, such as within 5 hours from the factor onset. Many of the
compounds exhibit a long term effect whereby administration of the
compounds may be conducted with long intervals, such as 1 week or 2
weeks.
In connection with the use in nerve guides, the administration may
be continuous or in small portions based upon controlled release of
the active compound(s). Furthermore, precursors may be used to
control the rate of release and/or site of release. Other kinds of
implants and well as oral administration may similarly be based
upon controlled release and/or the use of precursors.
As discussed above, the present invention relates to treatment of
individuals for inducing differentiation, modulating proliferation,
stimulate regeneration, neuronal plasticity and survival of cells
in vitro or in vivo, the treatment involving administering an
effective amount of one or more compounds as defined above.
Another strategy for administration is to implant or inject cells
capable of expressing and secreting the compound in question.
Thereby the compound may be produced at the location where it is
going to act.
Treatment
The compounds according to the invention are particularly useful
for treating inflammatory diseases and conditions. The compounds
are useful for the diseases and conditions mentioned below, in
particular useful for the treatment of inflammation in association
with Rheumatoid arthritis and autoimmune diseases, as well as with
Alzheimer's disease, Parkinson's disease and Huntington's
disease.
Examples of disorders associated with inflammation that can be
treated with the compounds of the invention include;
neuroinflammation, Alzheimer's disease, Parkinson's disease and
Huntington's disease, asthma and other allergic reactions,
autoimmune diseases such as Acute disseminated encephalomyelitis
(ADEM), Addison's disease, ALS, Ankylosing spondylitis,
Antiphospholipid antibody syndrome (APS), Autoimmune hemolytic
anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Bullous
pemphigoid, Coeliac disease, Chagas disease, Chronic obstructive
pulmonary disease, Dermatomyositis, Diabetes mellitus type 1,
Endometriosis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome (GBS), Hashimoto's disease, Hidradenitis
suppurativa, Idiopathic thrombocytopenic purpura, Interstitial
cystitis, Lupus erythematosus, Morphea, Multiple sclerosis,
Myasthenia gravis, Narcolepsy, Neuromyotonia, Pemphigus Vulgaris,
Pernicious anaemia, Polymyositis, Primary biliary cirrhosis,
Rheumatoid arthritis, Schizophrenia, Scleroderma, Sjogren's
syndrome, SLE, Temporal arteritis (also known as "giant cell
arteritis"), Vasculitis, Vitiligo, Wegener's granulomatosis;
chronic inflammation, chronic prostatitis, glomerulonephritis,
hypersensitivities, inflammatory bowel diseases, pelvic
inflammatory disease, reperfusion injury, rheumatoid arthritis,
transplant rejection, vasculitis, osteoarthritis, tendovaginitis,
and arthritis.
The treatment may also be of persistent acute inflammation due to
non-degradable pathogens, persistent foreign bodies, or autoimmune
reactions, inflammatory disease of the central nervous system, such
as meningitis, encephalitis, inflammatory and toxic neuropathy,
including acute infective polyneuritis, inflammatory disorders with
tissue damage, HIV, hepatitis, osteoarthritis, tendovaginitis, and
arthritis.
In one embodiment the treatment may be of non-immune diseases with
aetiological origins in inflammatory processes including cancer,
atherosclerosis, and ischaemic heart disease.
Antibody
It is an objective of the present invention to provide the use of
an antibody, antigen binding fragment or recombinant protein
thereof capable of selectively binding to an epitope comprising a
contiguous amino acid sequence derived from interleukin-4 or a
fragment, homologue or variant thereof. The invention relates to
any antibody capable of selectively binding to an epitope
comprising a contiguous amino acid sequence derived from
interleukin-4, selected from any of the sequences set forth in SEQ
ID NOS: 1-37, or a fragment or variant of said sequence.
By the term "epitope" is meant the specific group of atoms (on an
antigen molecule) that is recognized by (that antigen's)
antibodies. The term "epitope" is the equivalent to the term
"antigenic determinant". The epitope may comprise 3 or more amino
acid residues, such as for example 4, 5, 6, 7, 8 amino acid
residues, located in close proximity, such as within a contiguous
amino acid sequence, or located in distant parts of the amino acid
sequence of an antigen, but due to protein folding have been
approached to each other.
Antibody molecules belong to a family of plasma proteins called
immunoglobulins, whose basic building block, the immunoglobulin
fold or domain, is used in various forms in many molecules of the
immune system and other biological recognition systems. A typical
immunoglobulin has four polypeptide chains, containing an antigen
binding region known as a variable region and a non-varying region
known as the constant region.
Native antibodies and immunoglobulins are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies between the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (VH) followed by a
number of constant domains. Each light chain has a variable domain
at one end (VL) and a constant domain at its other end. The
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light and heavy chain variable domains (Novotny J,
& Haber E. Proc Natl Acad Sci USA. 82(14):4592-6, 1985).
Depending on the amino acid sequences of the constant domain of
their heavy chains, immunoglobulins can be assigned to different
classes. There are at least five (5) major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG-1,
IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chains constant
domains that correspond to the different classes of immunoglobulins
are called alpha (.alpha.), delta (.delta.), epsilon (.epsilon.),
gamma (.gamma.) and mu (.mu.), respectively. The light chains of
antibodies can be assigned to one of two clearly distinct types,
called kappa (.kappa.) and lambda (.lamda.), based on the amino
sequences of their constant domain. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
The term "variable" in the context of variable domain of
antibodies, refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies.
The variable domains are for binding and determine the specificity
of each particular antibody for its particular antigen. However,
the variability is not evenly distributed through the variable
domains of antibodies. It is concentrated in three segments called
complementarity determining regions (CDRs) also known as
hypervariable regions both in the light chain and the heavy chain
variable domains.
The more highly conserved portions of variable domains are called
the framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies. The constant domains are not involved directly in
binding an antibody to an antigen, but exhibit various effector
functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
An antibody that is contemplated for use in the present invention
thus can be in any of a variety of forms, including a whole
immunoglobulin, an antibody fragment such as Fv, Fab, and similar
fragments, a single chain antibody which includes the variable
domain complementarity determining regions (CDR), and the like
forms, all of which fall under the broad term "antibody", as used
herein. The present invention contemplates the use of any
specificity of an antibody, polyclonal or monoclonal, and is not
limited to antibodies that recognize and immunoreact with a
specific antigen. In the context of both the therapeutic and
screening methods described below, preferred embodiments are the
use of an antibody or fragment thereof that is immunospecific for
an antigen or epitope of the invention.
The term "antibody fragment" refers to a portion of a full-length
antibody, generally the antigen binding or variable region.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2 and
Fv fragments. Papain digestion of antibodies produces two identical
antigen binding fragments, called the Fab fragment, each with a
single antigen binding site, and a residual "Fc" fragment,
so-called for its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen binding
fragments that are capable of cross-linking antigen, and a residual
other fragment (which is termed pFc'). Additional fragments can
include diabodies, linear antibodies, single-chain antibody
molecules, and multispecific antibodies formed from antibody
fragments. As used herein, "functional fragment" with respect to
antibodies, refers to Fv, F(ab) and F(ab').sub.2 fragments.
The term "antibody fragment" is used herein interchangeably with
the term "antigen binding fragment".
Antibody fragments may be as small as about 4 amino acids, 5 amino
acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 amino
acids, about 15 amino acids, about 17 amino acids, about 18 amino
acids, about 20 amino acids, about 25 amino acids, about 30 amino
acids or more. In general, an antibody fragment of the invention
can have any upper size limit so long as it is has similar or
immunological properties relative to antibody that binds with
specificity to an epitope comprising a peptide sequence selected
from any of the sequences identified herein as SEQ ID NOs: 1-37, or
a fragment of said sequences. Thus, in context of the present
invention the term "antibody fragment" is identical to the term
"antigen binding fragment".
Antibody fragments retain some ability to selectively bind with its
antigen or receptor. Some types of antibody fragments are defined
as follows: (1) Fab is the fragment that contains a monovalent
antigen-binding fragment of an antibody molecule. A Fab fragment
can be produced by digestion of whole antibody with the enzyme
papain to yield an intact light chain and a portion of one heavy
chain. (2) Fab' is the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain. Two Fab' fragments are obtained per antibody
molecule.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region. (3)
(Fab').sub.2 is the fragment of an antibody that can be obtained by
treating whole antibody with the enzyme pepsin without subsequent
reduction. (4) F(ab').sub.2 is a dimer of two Fab' fragments held
together by two disulfide bonds.
Fv is the minimum antibody fragment that contains a complete
antigen recognition and binding site. This region consists of a
dimer of one heavy and one light chain variable domain in a tight,
non-covalent association (V.sub.H-V.sub.L dimer). It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen
binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding site.
(5) Single chain antibody ("SCA"), defined as a genetically
engineered molecule containing the variable region of the light
chain, the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain molecule.
Such single chain antibodies are also referred to as "single-chain
Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide
further comprises a polypeptide linker between the VH and VL
domains that enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies 113: 269-315 Rosenburg and
Moore eds. Springer-Verlag, N.Y., 1994.
The term "diabodies" refers to small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy chain
variable domain (VH) connected to a light chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161, and Hollinger et al., Proc. Natl. Acad Sci. USA 90:
6444-6448 (1993).
The invention also contemplates multivalent antibodies having at
least two binding domains. The binding domains may have specificity
for the same ligand or for different ligands. In one embodiment the
multispecific molecule is a bispecific antibody (BsAb), which
carries at least two different binding domains, at least one of
which is of antibody origin. Multivalent antibodies may be produced
by a number of methods. Various methods for preparing bi- or
multivalent antibodies are for example described in U.S. Pat. Nos.
5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;
5,013,653; 5,258,498; and 5,482,858.
The invention contemplate both polyclonal and monoclonal antibody,
antigen binding fragments and recombinant proteins thereof which
are capable of binding an epitope according to the invention.
The preparation of polyclonal antibodies is well-known to those
skilled in the art. See, for example, Green et al. 1992. Production
of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.),
pages 1-5 (Humana Press); Coligan, et al., Production of Polyclonal
Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols
in Immunology, section 2.4.1, which are hereby incorporated by
reference.
The preparation of monoclonal antibodies likewise is conventional.
See, for example, Kohler & Milstein, Nature, 256:495-7 (1975);
Coligan, et al., sections 2.5.1-2.6.7; and Harlow, et al., in:
Antibodies: A Laboratory Manual, page 726, Cold Spring Harbor Pub.
(1988), Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography. See, e.g., Coligan, et al., sections
2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al., Purification
of Immunoglobulin G (IgG). In: Methods in Molecular Biology, 1992,
10:79-104, Humana Press, N.Y.
Methods of in vitro and in vivo manipulation of monoclonal
antibodies are well known to those skilled in the art. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, 1975, Nature 256, 495-7, or may be made by
recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
The monoclonal antibodies for use with the present invention may
also be isolated from phage antibody libraries using the techniques
described in Clackson et al., 1991, Nature 352: 624-628, as well as
in Marks et al., 1991, J Mol Biol 222: 581-597. Another method
involves humanizing a monoclonal antibody by recombinant means to
generate antibodies containing human specific and recognizable
sequences. See, for review, Holmes, et al., 1997, J Immunol
158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma
& Immunol 81:105-115.
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
additional to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method.
The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity (U.S. Pat. No. 4,816,567); Morrison et al.,
1984, Proc Natl Acad Sci 81: 6851-6855.
Methods of making antibody fragments are also known in the art (see
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, NY, 1988, incorporated herein by
reference). Antibody fragments of the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab').sub.2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking
group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, in U.S. Pat. No. 4,036,945 and
U.S. Pat. No. 4,331,647, and references contained therein. These
patents are hereby incorporated in their entireties by
reference.
Other methods of cleaving antibodies, such as separation of heavy
chains to form monovalent light-heavy chain fragments, further
cleavage of fragments, or other enzymatic, chemical, or genetic
techniques may also be used, so long as the fragments bind to the
antigen that is recognized by the intact antibody. For example, Fv
fragments comprise an association of V.sub.H and V.sub.L chains.
This association may be noncovalent or the variable chains can be
linked by an intermolecular disulfide bond or cross-linked by
chemicals such as glutaraldehyde. Preferably, the Fv fragments
comprise V.sub.H and V.sub.L chains connected by a peptide linker.
These single-chain antigen binding proteins (sFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow, et al., 1991, In:
Methods: A Companion to Methods in Enzymology, 2:97; Bird et al.,
1988, Science 242:423-426; U.S. Pat. No. 4,946,778; and Pack, et
al., 1993, BioTechnology 11:1271-77.
Another form of an antibody fragment is a peptide coding for a
single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") are often involved in antigen
recognition and binding. CDR peptides can be obtained by cloning or
constructing genes encoding the CDR of an antibody of interest.
Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of
antibody-producing cells. See, for example, Larrick, et al.,
Methods: a Companion to Methods in Enzymology, Vol. 2, page 106
(1991).
The invention contemplates human and humanized forms of non-human
(e.g. murine) antibodies. Such humanized antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) that contain a minimal sequence derived
from non-human immunoglobulin, such as the epitope recognising
sequence. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a nonhuman species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. Humanized antibody(es)
containing a minimal sequence(s) of antibody(es) of the invention,
such as a sequence(s) recognising an epitope(s) described herein,
is one of the preferred embodiments of the invention.
In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and optimize antibody performance. In general, humanized antibodies
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see: Jones et al., 1986, Nature 321, 522-525; Reichmann et
al., 1988, Nature 332, 323-329; Presta, 1992, Curr Op Struct Biol
2:593-596; Holmes et al., 1997, J Immunol 158:2192-2201 and
Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol
81:105-115.
The generation of antibodies may be achieved by any standard
methods in the art for producing polyclonal and monoclonal
antibodies using natural or recombinant fragments of a sequence
selected from any of the sequences identified as SEQ ID NOs: 1-37,
as an antigen. Such antibodies may be also generated using variants
or fragments of SEQ ID NOs: 1-37.
The antibodies may also be produced in vivo by the individual to be
treated, for example, by administering an immunogenic fragment
according to the invention to said individual. Accordingly, the
present invention further relates to a vaccine comprising an
immunogenic fragment described above.
The application also relates to a method for producing an antibody
of the invention said method comprising a step of providing of an
immunogenic fragment described above.
The invention relates both to an antibody, which is capable of
modulating, such as enhancing or attenuating, biological function
of IL-4 in particular a function related to inflammation, and to an
antibody, which can recognise and specifically bind to IL-4 without
modulating biological activity thereof.
The invention relates to use of the above antibodies for
therapeutic applications involving the modulation of activity of
IL-4.
In one aspect the invention relates to the use of a pharmaceutical
composition comprising an antibody described above.
EXAMPLES
Example 1
Four peptides derived from IL-4 were designed and synthesized (SEQ
ID NOs:1-4). Mapping of the location of the peptides was performed
employing PyMOL.TM. software, based on PyMOL v0.99 (DeLano
Scientific LLC, South San Francisco, Calif., U.S.A). This was done
based on the crystal structure of the ternary complex of human
Il4-Il4r-Il13ra, PDB ID: 3BPN and 3BPL (LaPorte et al., 2008).
IL-4 interacts with two fibronectin type III modules (FN3-1 and
FN3-2) of the extracellular part of the IL-4R.alpha.) (FIGS. 1 and
2). IL-4 interacts with two fibronectin type III modules (FN3-1 and
FN3-2) of the extracellular part of IL-4R.alpha. and .gamma.c
(FIGS. 3 and 4).
Example 2
4 peptides derived from IL-4 were tested in a neurite outgrowth
assay whether they had any biological activity.
Cerebellar granular neurons (CGN) were prepared from 3 or 7
postnatal (P) day Wistar rats (Charles River, Sulzfeld, Germany or
Taconic, Ejby, Denmark). Cerebella were cleared of meninges and
blood vessels, roughly homogenized by chopping, and trypsinized
with trypsin from Sigma-Aldrich (Brondby, Denmark). The neurons
were washed in the presence of DNAse 1 and soybean trypsin
inhibitor (Sigma-Aldrich), and cellular debris was pelleted by
centrifugation before plating. For single-cell culture experiments,
P7 CGNs were plated at a density of 10,000 cells/well onto uncoated
eight-well Lab-Tek chamber slides (NUNC, Slangerup, Denmark) in
Neurobasal-A medium supplemented with 0.4% (w/v) BSA. Peptides at
various concentrations were added to the medium immediately after
plating, and cells were maintained at 37.degree. C. and 5% CO.sub.2
for 24 h. Cultures then were fixed, blocked and incubated with
polyclonal rabbit antibody against rat GAP-43 (Chemicon, Temecula,
Calif., USA) followed by incubation with secondary Alexa Fluor488
goat anti-rabbit antibody (Molecular Probes, Eugene, Oreg., USA) as
previously described (Neiiendam et al., 2004). The immunostained
cultures were all recorded by computer-assisted fluorescence
microscopy using a Nikon Diaphot inverted microscope (Nikon, Japan)
equipped with a Nikon Plane 20.times. objective. Images were
captured with a charge-coupled device video camera (Grundig
Electronics, Nurnberg, Germany) using the software package Prima
developed at the Protein Laboratory (University of Copenhagen,
Copenhagen, Denmark). The length of neuronal processes per cell was
estimated using the software package Process Length developed at
the Protein Laboratory (Ronn et al. 2000). For estimation of
neurite outgrowth, at least 200.+-.20 cells were processed for each
group in each individual experiment.
Results:
Peptides with the SEQ ID NOs: 1, 2, 3, and 4, from the IL-4 binding
site were found to induce a neuritogenic response from primary
neurons. The results of the effect of SEQ ID NO:1 2, 3 and 4 on
cerebellar neurite outgrowth are shown in FIGS. 5, 6, 9, and 12,
respectively.
Example 3
Primary macrophage cells (or cells of the AMJ2C8 macrophage cell
line, see Ryan et al., 1997) can be cultured for 24 h at a density
of 6.times.10.sup.-5 cells/ml in 12-well plates (Nunc, Slangerup,
Denmark) at 37.degree. C., in 5% CO.sub.2 and 95% humidity. For
determination of TNF-.alpha. release in response to LPS
stimulation, triplicate cultures were cultured in DMEM with 10% FCS
for 24 h and then stimulated with 0-10 .mu.g/ml LPS for an
additional 24 h period, after which culture supernatants were
collected. Determination of TNF-.alpha. concentrations in
conditioned media from LPS-treated macrophages was done employing
the L929 fibroblast-like cells which were sensitive to TNF-.alpha.
upon exposure to actinomycin D (He et al., 2002). L929 cells were
seeded in 96-well plates at a density of 20.000 cells per well and
maintained at 37.degree. C., 5% CO.sub.2, RPMI 1640 supplemented
with 10% FCS and 0.5% penicillin-streptomycin. At 1 h prior to use
as the TNF-.alpha. bioassay, L929 cells were pre-treated with 5
.mu.g/ml actrinomycin D (Sigma), and further incubated with
conditioned medium, in various dilutions, from LPS-treated
macrophage cultures. Cell viability was than evaluated using the
CellTiter 96 assay (Promega, Madison, Wis., USA).
Macrophage Activation Test-System
Macrophages were seeded in 6 well multidish with 9.6 cm.sup.2 per
well, in the density 10.000 cells/well. Peptides or protein with
potential anti-inflammatory effects were added to the culture. As
negative control, medium was added to one well and as a positive
control, 100 .mu.M hydrocortisone was added to one well. Cell
cultures were incubated for 24 h at 37.degree. C. IFN-.gamma. was
added to the macrophage cultures to activate macrophages in the
concentration 0.01 .mu.g/ml. As control no IFN-.gamma. but medium
was added to one well. Fibroblast cells were seeded in a 96 well
plate, in the concentration 0.2.times.10.sup.2 cells/ml. Both cell
cultures were incubated for 24 h at 37.degree. C.
Conditioned medium from macrophages was collected by spinning the
cell solution for 5 min at 1200 rpm. The conditioned medium was
added to fibroblasts, TNF-.alpha. was added for the titration curve
and finally actinomycin D was added to the fibroblasts in the
concentration 0.5 .mu.g/ml.
Results:
The effect of peptides with SEQ ID NOs:1, 3, 5, 6, and 19 on
inhibition of an inflammatory response in macrophage cell cultures
was tested. Results are shown in FIGS. 7, 10, and 13-17.
Example 4
Binding Studies Using Surface Plasmon Resonance (SPR) Analysis
Recombinant IL4R.alpha. was immobilized on a CM5 sensor chip. The
immobilization process was done by activating the carboxymethylated
dextran matrix with 35 .mu.l activation solution followed by an
injection of protein in 10 mM sodium acetate solution (pH 5.0).
After a desired level of protein was immobilized 35 .mu.l of
deactivation solution is injected to deactivate any free
carboxymethylated groups in the dextran matrix. One flow cell was
always empty as a control. Each analyte (recombinant IL-4 or
IL-4-derived peptides) was diluted in PBS and injected at a flow
rate of 10 .mu.l/min. The obtained data was analyzed by performing
a non-linear curve fitting using the software BIAevaluation v.4
from Biacore. The curves were fitted to a 1:1 Langmuir binding
model which describes the interaction of two molecules in 1:1
complex. The affinity constant (K.sub.D) was calculated from the
association rate constant (k.sub.a) and the dissociation rate
constant (k.sub.d). This was done by using the following formula,
where L is the immobilized ligand, A the analyte, and LA is the
analyte-ligand complex:
.times..times..times..times..times..times..times..times..times..fwdarw..r-
arw..times..times..times. ##EQU00001##
Rate of Decreasing Ligand Concentration
dd.times..times. ##EQU00002##
Rate of Increasing Product Concentration
d.times..times.d.times..times. ##EQU00003##
At Steady State
d.times..times.d.times..times..times..times..times..times.
##EQU00004## Results:
Binding between Ph2 (SEQ ID NO:3), and IL4r.alpha., and between Ph3
(SEQ ID NO:1) and IL4r.alpha. was studied. The results are shown in
FIGS. 8 and 11, respectively.
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structure of the interleukin-4/receptor .alpha. chain complex
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and nuclear magnetic resonance models. 1995, 247, 360-372.
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FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth
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Berezin A, Berezin V, Moller A, Bock, E A simple procedure for
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SEQUENCE LISTINGS
1
38116PRTArtificial SequenceVariant of fragment of interleukin-4
1Ala Gln Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg Ala1 5
10 15214PRTArtificial SequenceVariant of fragment of interleukin-4
2Ala Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser Ala1 5
10314PRTArtificial SequenceVariant of fragment of interleukin-4
3Ala Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Gly1 5
10415PRTArtificial SequenceVariant of fragment of interleukin-4
4Ala Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Ser1 5 10
1558PRTArtificial SequenceVariant of fragment of interleukin-4 5Leu
Gln Glu Ile Lys Thr Leu Asn1 5610PRTArtificial SequenceVariant of
fragment of interleukin-4 6Lys Arg Leu Gln Gln Asn Leu Phe Gly Gly1
5 10718PRTArtificial SequenceVariant of fragment of interleukin-4
7Ala Cys Ala Gln Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys1 5
10 15Arg Ala87PRTArtificial SequenceVariant of fragment of
interleukin-4 8Gln Glu Ile Ile Lys Lys Leu1 5913PRTArtificial
SequenceVariant of fragment of interleukin-4 9Ala Ile Gln Asn Gln
Glu Glu Ile Lys Tyr Leu Asn Ser1 5 10107PRTArtificial
SequenceVariant of fragment of interleukin-4 10Ala Ile Ile Leu Gln
Glu Ile1 5117PRTArtificial SequenceVariant of fragment of
interleukin-4 11Ile Val Leu Gln Glu Ile Ile1 51211PRTArtificial
SequenceVariant of fragment of interleukin-4 12Thr Leu Gly Glu Ile
Ile Lys Gly Val Asn Ser1 5 101315PRTArtificial SequenceVariant of
fragment of interleukin-4 13Val Thr Leu Ile Asp His Ser Glu Glu Ile
Phe Lys Thr Leu Asn1 5 10 15149PRTArtificial SequenceVariant of
fragment of interleukin-4 14Leu Gln Glu Arg Ile Lys Ser Leu Asn1
51513PRTArtificial SequenceVariant of fragment of interleukin-4
15Arg Leu Asp Arg Glu Asn Val Ala Val Tyr Asn Leu Trp1 5
10168PRTArtificial SequenceVariant of fragment of interleukin-4
16Leu Arg Ser Leu Asp Arg Asn Leu1 5178PRTArtificial
SequenceVariant of fragment of interleukin-4 17Arg Leu Leu Arg Leu
Asp Arg Asn1 51814PRTArtificial SequenceVariant of fragment of
interleukin-4 18Arg Phe Leu Lys Arg Tyr Phe Tyr Asn Leu Glu Glu Asn
Leu1 5 101915PRTArtificial SequenceVariant of fragment of
interleukin-4 19Arg Asn Lys Gln Val Ile Asp Ser Leu Ala Lys Phe Leu
Lys Arg1 5 10 15207PRTArtificial SequenceVariant of fragment of
interleukin-4 20Arg His Lys Ala Leu Ile Arg1 5218PRTArtificial
SequenceVariant of fragment of interleukin-4 21Lys Lys Leu Ile Arg
Tyr Leu Lys1 5227PRTArtificial SequenceVariant of fragment of
interleukin-4 22Arg His Lys Thr Leu Ile Arg1 5238PRTArtificial
SequenceVariant of fragment of interleukin-4 23Met Gln Asp Lys Tyr
Ser Lys Ser1 52415PRTArtificial SequenceVariant of fragment of
interleukin-4 24Ala Glu Arg Val Lys Ile Glu Gln Arg Glu Tyr Lys Lys
Tyr Ser1 5 10 152511PRTArtificial SequenceVariant of fragment of
interleukin-4 25Ser Gln Leu Ile Arg Phe Leu Lys Arg Leu Ala1 5
102613PRTArtificial SequenceVariant of fragment of interleukin-4
26Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr1 5
102712PRTArtificial SequenceVariant of fragment of interleukin-4
27Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ala1 5
102812PRTArtificial SequenceVariant of fragment of interleukin-4
28Thr Glu Lys Glu Val Leu Arg Gln Phe Tyr Ser Ala1 5
102912PRTArtificial SequenceVariant of fragment of interleukin-4
29Lys Thr Leu Thr Glu Leu Thr Lys Thr Leu Asn Ser1 5
103015PRTArtificial SequenceVariant of fragment of interleukin-4
30Ala His Lys Glu Ile Ile Lys Thr Leu Asn Ser Leu Gln Lys Ala1 5 10
153112PRTArtificial SequenceVariant of fragment of interleukin-4
31Ala Lys Thr Leu Ser Thr Glu Leu Thr Val Thr Ala1 5
103211PRTArtificial SequenceVariant of fragment of interleukin-4
32Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Ala1 5
103311PRTArtificial SequenceVariant of fragment of interleukin-4
33Asn Glu Glu Arg Leu Lys Thr Ile Met Arg Ala1 5
103412PRTArtificial SequenceVariant of fragment of interleukin-4
34Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser Arg1 5
103511PRTArtificial SequenceVariant of fragment of interleukin-4
35Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys Thr1 5
103610PRTArtificial SequenceVariant of fragment of interleukin-4
36Ala His Arg His Lys Gln Leu Ile Arg Ala1 5 103712PRTArtificial
SequenceVariant of fragment of interleukin-4 37Ala Thr Ala Gln Gln
Phe His Arg His Lys Gln Ala1 5 1038153PRTHumanMISC_FEATUREHuman
interleukin-4 38Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe
Leu Leu Ala1 5 10 15Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp
Ile Thr Leu Gln 20 25 30Glu Ile Ile Lys Thr Leu Asn Ser Leu Thr Glu
Gln Lys Thr Leu Cys 35 40 45Thr Glu Leu Thr Val Thr Asp Ile Phe Ala
Ala Ser Lys Asn Thr Thr 50 55 60Glu Lys Glu Thr Phe Cys Arg Ala Ala
Thr Val Leu Arg Gln Phe Tyr65 70 75 80Ser His His Glu Lys Asp Thr
Arg Cys Leu Gly Ala Thr Ala Gln Gln 85 90 95Phe His Arg His Lys Gln
Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg 100 105 110Asn Leu Trp Gly
Leu Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala 115 120 125Asn Gln
Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met 130 135
140Arg Glu Lys Tyr Ser Lys Cys Ser Ser145 150
* * * * *